Title: Intromission Pattern and Species Vaginal Code in Relation to Induction of Pseudopregnancy

Author: Milton Diamond Ph.D.

Published in: Science, 4 September 1970, Volume 169, pp. 995-997

Abstract

Mechanical stimulation was used to mimic normal vaginal stimuli during copulation in the mouse; the number and rate of intromissions were crucial influences on reproductive success. The best combinations for the mechanical induct ion of pseudopregnancy were comparable to those male behaviors normally seen during mating. The behavior of the male coincides with a species-related vaginal code, increasing chances for reproductive success between cospecifics.

Observation of the copulatory behavior of many species, rodents in particular, reveals a definite series of intro- missions interspersed with other sexual or nonsexual behaviors. With the report1 indicating that the induction of pregnancy and pseudopregnancy in the hamster is related to the number and rate of intromissions, the hypothesis was formulated that the proper combination of the two would be crucial for successful reproduction in other species. Appreciation of such interacting factors would amplify the data for the rat. For the rat, Ball indicated that the number of intromissions per se is inconsequential for pregnancy as long as at least one or two ejaculations occur (evidenced by the finding of sperm plugs)2. More recently, four or more intromissions, rather than three or fewer, are reported to significantly increase chances of pregnancy3.

I have studied the combined effect of number and frequency of intromissions (insertions and interval between insertions) by means of mechanical induction of pseudopregnancy in the mouse. Heretofore, the induction of pseudopregnancy in this species has been impossible or rare without the use of an ejaculating vasectomized male4-6.

Virgin DBA/2J strain mice obtained from the Jackson Laboratories (Bar Harbor, Maine) or bred in our laboratory were used. This strain was chosen because its copulatory behavior has been extensively studied7.

All females were given freshly prepared ovulation-inducing gonadotropins. Pregnant mares’ serum (PMS) [Equinex (Ayerst), serum gonadotropin; 2 international units] was administered at 4:30 to 5:30 p.m. followed 48 hours later by 3 units of human chorionic gonadotropin (HCG) [“A.P.L.” (Ayerst), chorionic gonadotropin]. Between 9:00 and 11:00 a.m. on the day after administration of HCG, the females were vaginally stimulated so as to simulate intromission. Stimulation was with a mechanical vibrator (Vibro-Graver, Burgess Vibro Crafters, Inc., Chicago) fitted with a polished brass “penis” 3.5 mm in diameter. The penis rapidly moved back and forth (7200 strokes per minute) and was inserted up to the cervix. The day after stimulation was considered day 1. Until stimulation, the test females were kept in the colony room. After stimulation, the females were kept in a separate room without males to forestall pheromonal effects.

Table 1. Mechanical induction of pseudopregnancy in the DBA/2J mouse. Females were judged pseudopregnant if the traumatized uterine horn weighed more than 90 mg and more than 150 percent of the weight of the control. Abbreviations: I, number of vaginal insertions; II, duration of each insertion; III, interval between insertions; TIT, total insertion time; and TET, total duration of experimental exposure.

I
(No.)

II
(sec)

III
(sec)

TIT
(sec)

TET
(sec)

Females made
pseudo- pregnant
(No.)

Females tested
(No.)

Success
(%)

1
1
1
1
1

20
50
100
200
300

0
0
0
0
0

20
50
100
200
300

20
50
100
200
300

2
1
2
4
4

14
6
7
9
8

14
17
29
44
50

3
3
3
3
3

20
20
20
20
20

30
60
120
180
270

60
60
60
60
60

150
240
420
600
870

6
2
4
2
3

8
6
10
6
6

75
33
40
33
50

5
5
5
5
5

20
20
20
20
20

30
60
120
180
270

100
100
100
100
100

250
400
700
1000
1450

4
2
4
2
5

7
8
10
7
8

58
25
40
28
63

10
10
10
10
10

20
20
20
20
20

30
60
120
180
270

200
200
200
200
200

500
800
1400
2000
2900

1
4
12
5
2

6
9
17
11
7

17
44
70
45
29

15
15
15
15
15

20
20
20
20
20

30
60
120
180
270

300
300
300
300
300

750
1200
2100
3000
4350

3
3
3
2
5

7
7
11
8
6

43
43
27
25
87

30
30

5
15

5
45

150
450

300
1800

5
2

17
3

29
67

Various aspects of stimulation were tested (Table 1). The number (I) of insertions used was 3, 5, 10, or 15; the intervals between insertions (III) varied from 30 to 270 seconds. The median duration of intromission for males of this strain varies from 17 to 20 seconds; therefore, duration of each insertion (II) was standardized to last 20 seconds. The selection of these values was predicated on the extensive behavioral studies by McGill and his colleagues5,7. The comparative efficiency of single prolonged stimuli and several other aspects of stimulation were also tested.

The effectiveness of stimuli in the induction of pseudopregnancy was evaluated in every case by a modification of the uterine decidual reaction technique8. One uterine horn was traumatized by being extensively cut along the antimesometrial wall 3 days after stimulation (day 3); it was checked for a decidual reaction 3 days later (day 6). A decidual reaction was said to have occurred if the traumatized uterine horn weighed at least 50 percent more than the untraumatized horn and at least 90.0 mg (mean weight of the untraumatized control horn was 47.3 mg). Occasionally, ovaries were examined histologically for the prolonged maintenance of corpora lutea necessary for pregnancy and pseudopregnancy.

Throughout the experiment the mice were grouped one to four per clear plastic container (approximately 28 by 18 by 12 cm) with wood-chip bedding. The light cycle was maintained with darkness occurring from 6 p.m. to 6 a.m. Food and water were available as desired. The room was air-conditioned; temperatures varied from 65° to 75°F (18.3° to 24°C).

Mice can be mechanically induced to pseudopregnancy (Table 1). Consistent with our findings for the hamster (I), neither many insertions nor a single prolonged stimulation alone will insure the induction of pseudopregnancy. The successful stimulatory patterns appear to represent a key to a vaginal code which, within certain limits, is relatively specific for each species. Patterns of stimulation optimum for the induction of pseudopregnancy in the hamster1 (I = 30, II = 5, III = 5) or capable of inducing 100 percent pseudopregnancy in the rat9 (I = 1, II = 20) are relatively ineffective in the mouse. The pattern of stimulation sufficient to induce pseudopregnancy in the rat had been reported ineffective in the hamster and mouse4. For the mouse, as for the hamster, the combination of factors most successful are those which resemble the normal mating pattern. McGill and colleagues5,7 have reported that the median number of intromissions for this strain of mice varied in different studies from 5 to 13 and the median time between intromissions approximated 90 to 215 seconds. I find a wide range of stimuli with patterns approximating these capable of effectively inducing the changes characteristic of pregnancy. For example, ten stimuli applied at 120-second intervals and 15 stimuli applied at 270-second intervals are highly effective in initiating pseudo-pregnancy10. Significantly, a similarly long interval between intromissions is comparable to that seen in the normally copulating male.

In the variation of insertion number and the interval between insertions, the total insertion time (TIT) of vaginal stimulation and the total time of experimental exposure (TET) were also varied. These factors are comparable to the total intromission intervals and the total period allowed for mating. As seen in the hamster1, prolongation of a single vaginal stimulation is not an efficient way to initiate pseudopregnancy. The maximum stimulus tested, of a duration comparable to the largest TIT (300 seconds), is not significantly more effective than shorter TIT’s if the latter is the sum of spaced periodic insertions. The duration of the individual insertions must be considered too; even thirty 5-second insertions (TIT = 150) evenly spaced throughout 300 seconds (TIT = 5) were not too effective in inducing pseudopregnancy. The TET is also crucial; with multiple insertions, longer exposures seem better. These are comparable to the long median latencies to ejaculation (from 1376 to 1946 seconds) reported for this strain5,7.

Naturally, normal mating is not structured so that only one pattern can insure initiation of pregnancy; indeed a wide combination of insertion numbers and intervals between insertions will suffice to induce maintenance of corpora lutea (Table 1). However, those stimulations that include multiple intromissions, that have more than 5 seconds between insertions, or that have extended exposure time will be relatively successful in inducing gestational changes. An increase in the number of insertions, the interval between insertions, or the TET will generally increase the chance for the initiation of pseudopregnancy until an optimum range of combinations is reached. Extremes of stimulation, however, do not necessarily increase the chances for induction of pseudopregnancy. Significantly, the so-called “vaginal code” seems flexible and depends upon the interaction between several independent variables among which are insertion number, rate of insertion, total exposure time, and duration of each insertion. Apparently some neural “comparator” or “integrator” is involved in the integration of these variables to affect the neuroendocrine reflexes. Undoubtedly other factors yet undefined are also integrated in a normal mating situation1.

Since the successful patterns of copulatory-like stimuli used here were based upon behavioral norms for this genetic strain, our success might be considered predictable; similarly consistent is the lack of success in other studies4 in which were used vaginal stimuli which mimic the copulatory patterns of other species and which differ markedly from those of the mouse.

Land and McGill5 have reported that in the mouse a large number of preejaculatory thrusts is neither necessary nor sufficient to induce luteal activity in the female. The ejaculatory reflex regardless of the number of thrusts, however, was considered crucial for the induction of pseudopregnancy6. I would extend and modify these interpretations so that the number of intromissions be considered as a variable to be weighed. Furthermore, I would emphasize that, given sufficient stimulation appropriate to the species-related vaginal code, the ejaculatory reflex would not be necessary for the initiation of mechanisms which induce pseudopregnancy, such as maintained corpora lutea. This consideration is crucial for understanding mechanisms for luteotropin release and for studying artificial insemination in mice. If the definition of pseudopregnancy given by Land and McGill5 is restated to encompass any prolongation of a normal 4- or 5-day ovarian cycle, even their data might be reinterpreted to show that 30 percent of 43 females were induced to pseudopregnancy by nonejaculating copulating males. My figures are then in keeping with theirs.

The stereotype in copulatory behavior seen in most animal species is hereby given significance. My results indicate that, within broad limits, a proper patterning of vaginal stimuli will induce maintenance of corpora lutea capable of supporting pregnancy.

Lincoln11 has recently reported that in the rat there are specific hypothalamic neurons which receive sensory projections from areas apparently located in the region of the cervix and which are specific in their response to cervical probing. Aron et al. and Zarrow et al.12 have demonstrated the quantitative interaction of copulatory stimuli and ovulation mediated by gonadotropins. Adler13 in a similar manner has demonstrated a relation between the number of intromissions and the probability of pseudopregnancy, and he, as well as Chester and Zucker14, have shown that copulatory behavior is related to sperm transport and pseudopregnancy in the rat. These authors call attention to the need for comparative data. Studies of the hamster further indicate that release of progestin initiated by particular mating stimuli may be involved15.

The concept is thus established that species-related neural stimuli from the vagina, integrated with other sensory inputs accompanying mating, are crucial for the initiation of neural and endocrine mechanisms supporting pregnancy and pseudopregnancy.

Note added in proof: Since preparation of this manuscript, McGill and Coughlin have suggested that the penile swelling of the ejaculating male leads to mechanical stretching of the vagina or cervix (or both) and that this is the stimulus which induces luteotropin release in the mouse16.

17. Supported in part by funds from the Ford Foundation, and by PHS grant HD 02066. The author is in part supported with a
Lederle Medical Faculty Award. I thank Miss Jennifer Wigington and Mrs. E. Barbara Zegart for their cooperation in the research.